The development of tetrazole derivatives as protein arginine methyltransferase I (PRMT I) inhibitors

Article abstract of DOI:10.3390/ijms20153840

Protein arginine methyltransferase 1 (PRMT1) can catalyze protein arginine methylation by transferring the methyl group from S-adenosyl-L-methionine (SAM) to the guanidyl nitrogen atom of protein arginine, which influences a variety of biological processes. The dysregulation of PRMT1 is involved in a diverse range of diseases, including cancer. Therefore, there is an urgent need to develop novel and potent PRMT1 inhibitors. In the current manuscript, a series of 1-substituted 1H-tetrazole derivatives were designed and synthesized by targeting at the substrate arginine-binding site on PRMT1, and five compounds demonstrated significant inhibitory effects against PRMT1. The most potent PRMT1 inhibitor, compound 9a, displayed non-competitive pattern with respect to either SAM or substrate arginine, and showed the strong selectivity to PRMT1 compared to PRMT5, which belongs to the type II PRMT family. It was observed that the compound 9a inhibited the functions of PRMT1 and relative factors within this pathway, and down-regulated the canonical Wnt/β-catenin signaling pathway. The binding of compound 9a to PRMT1 was carefully analyzed by using molecular dynamic simulations and binding free energy calculations. These studies demonstrate that 9a was a potent PRMT1 inhibitor, which could be used as lead compound for further drug discovery.

Full text of DOI:10.3390/ijms20153840

International Journal of
Molecular Sciences
Article
The Development of Tetrazole Derivatives as Protein
Arginine Methyltransferase I (PRMT I) Inhibitors
Yutong Sun 1,†, Zhe Wang 1,†, Hao Yang 1,†, Xuanli Zhu 1, Han Wu 1, Lu Ma 2, Fang Xu 3,
Wei Hong 4,5,
*
and Hao Wang 1,
*
1
School of Pharmacy, Ningxia Medical University, Yinchuan 750004, China
School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750004, China
School of Postgraduate Education, Ningxia Medical University, Yinchuan 750004, China
School of Chemistry and Chemical Engineering, North Minzu University, Yinchuan 750021, China
Key Laboratory of Chemical Engineering and Technology, State Ethnic Affairs Commission, North Minzu
University, Yinchuan 750021, China
2
3
4
5
*
Correspondence: hongwei136@hotmail.com (W.H.); paxhw@yahoo.co.uk (H.W.);
Tel.: +86-135-1920-2157 (W.H.); +86-135-1929-8012 (H.W.)
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 27 June 2019; Accepted: 1 August 2019; Published: 6 August 2019
Abstract: Protein arginine methyltransferase 1 (PRMT1) can catalyze protein arginine methylation by
transferring the methyl group from S-adenosyl-L-methionine (SAM) to the guanidyl nitrogen atom of
protein arginine, which influences a variety of biological processes. The dysregulation of PRMT1 is
involved in a diverse range of diseases, including cancer. Therefore, there is an urgent need to develop
novel and potent PRMT1 inhibitors. In the current manuscript, a series of 1-substituted 1H-tetrazole
derivatives were designed and synthesized by targeting at the substrate arginine-binding site on
PRMT1, and five compounds demonstrated significant inhibitory effects against PRMT1. The most
potent PRMT1 inhibitor, compound 9a, displayed non-competitive pattern with respect to either
SAM or substrate arginine, and showed the strong selectivity to PRMT1 compared to PRMT5, which
belongs to the type II PRMT family. It was observed that the compound 9a inhibited the functions of
PRMT1 and relative factors within this pathway, and down-regulated the canonical Wnt/β-catenin
signaling pathway. The binding of compound 9a to PRMT1 was carefully analyzed by using molecular
dynamic simulations and binding free energy calculations. These studies demonstrate that 9a was a
potent PRMT1 inhibitor, which could be used as lead compound for further drug discovery.
Keywords: PRMT1; structure-activity relationship (SAR); MOA studies; molecular dynamic
simulations; binding mode
1. Introduction
Protein arginine methyltransferases (PRMTs) transfer the methyl group from S-adenosyl-L-
methionine (SAM) to the guanidyl nitrogen atom of protein arginine, forming monomethylarginine
(mMA) and cofactor S-adenosyl-L-homocysteine (SAH) [
arginine methylation and influence a wide range of cellular processes, including transcriptional
regulation, RNA splicing, cell growth and differentiation [ ]. According to their different biological
characteristics, PRMTs are classified in three categories (Figure 1) [ ]. Type PRMTs (PRMT1, 2, 3, 4, 6
and 8) catalyze mono- and asymmetrical dimethylarginine (ADMA) of arginine residues, whereas,
1–3]. Therefore, PRMTs can catalyze protein
4,5
6
I
Type II PRMTs (PRMT5 and 9) catalyze mono- and symmetrical dimethylarginine (SDMA) [
7]. As the
only known Type III PRMT, PRMT7 catalyzes monomethylation of arginine [8].
Int. J. Mol. Sci. 2019, 20, 3840; doi:10.3390/ijms20153840
www.mdpi.com/journal/ijms

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Figure 1. Methylation states of protein arginine through protein arginine methyltransferases (PRMTs).
Previous studies showed that over 90% of the arginine methylation in mammalian cells was
produced by PRMT1 catalysis [9]. Furthermore, the disordered expression of PRMT1 is associated
with a variety of human diseases including pulmonary fibrosis [10], cardiovascular diseases [11],
diabetes, renal diseases [12] and cancer in particular [13–16]. Overexpression of PRMT1 can promote
the growth, survival, metastasis and invasion of tumor cells [17]. Therefore, PRMT1 is considered
to be a potential target for the treatment of cancer. Currently, a number of PRMT1 inhibitors have
been reported (Figure 2). In 2004, AMI-1 was found through high-throughput screening as the first
compound which displayed selectivity to PRMTs over protein lysine methyltransferases (PKMTs) [18].
Later, a number of PRMT1 inhibitors were reported, including RM65 [19], Stilbamidine [20], DB75 [21],
Allantodapsone [20] and DCLX069 [22]. However, the weak potency against PRMT1 limited their
therapeutic applications. Therefore, there is an urgent need to discover potent PRMT1 inhibitors.
Figure 2. The chemical structures of the typical reported PRMT1 inhibitors.
In the current study, a series of 1-substituted 1H-tetrazole derivatives were designed and
synthesized by targeting at the substrate arginine-binding site on PRMT1, and compound 9a was
discovered to be a potent PRMT1 inhibitor, which contained strong abilities to down-regulate
Wnt/β-catenin signaling pathway. Therefore, we suggest that compound 9a could be an important
lead compound for future PRMT-guided drug discovery.
2. Results and Discussion
2.1. The Molecular Design
The crystal structure of human PRMT1 (hPRMT1) has not yet been solved, so we used our
previously published homology model [23] in the current study. As shown in Figure 3A, the active site

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of PRMT1 is composed of the SAM and substrate arginine binding sites. In theory, if a compound can
bind to any sub-site, the methylation will be disturbed. In the current study, we used the arginine
binding site as a focus with which to design novel inhibitors. As Figure 3B shows, there are three amino
acids with aromatic rings (Tyr47, Tyr156 and Tyr160) that form a hydrophobic pocket, so we designed
two aromatic rings to form
as AR1 and AR2. Two negative charged residues Glu152 and Glu161 were reported to form strong
interactions with substrate arginine [24 25], so two positive charged centers were designed, which were
π-π and hydrophobic interactions with them, which were represented
,
represented as PC1 and PC2. In our previous publication [24], a group of compounds with central core
of phenyl (AR1) substituted furan (AR2) were discovered as PRMT1 inhibitors. In the current study,
we used the tetrazole ring to replace the furan ring as the bioisosteres for designing novel PRMT1
inhibitors. Moreover, in order to investigate the linker length between PC1 and PC2, the different
linking groups were incorporated, such as ethyl, propyl, piperazine. According to the pharmacophores
and analysis, a series of 1-phenyl substituted tetrazole derivatives (Figure 3C) were designed, in which
the benzene and tetrazole ring were according to AR1 and AR2 respectively, and the tertiary amine
and secondary amine were according to the PC1 and PC2, respectively.
Figure 3.
(SAH) and substrate arginine (basic regions are colored in gray, acidic regions are colored in ochre,
polar regions are colored in orange and nonpolar regions are colored in cyan); ( ) The pharmacophores
(A) The active site of human PRMT1 (hPRMT1) in complex with S-adenosyl-L-homocysteine
B
were designed based on the substrate arginine binding site. (AR = aromatic ring and PC = positive
center); (C) a series of tetrazole derivatives were designed based on the pharmacophores.
2.2. Chemistry
A series of 1,5-substituded tetrazole derivatives 9a–f, 10a–e, 16a–e, 18a–e and 20 were synthesized
as illustrated in Schemes 1–3. In Scheme 1, the commercially available substituted anilines 1a–f were
respectively reacted with ethyl oxalyl monochloride in the presence of triethylamine in anhydrous
dichloromethane to generate the compounds 2a
triphenylphosphine under refluxing in carbon tetrachloride gave compounds 3a
directly with sodium azide in acetonitrile to generate 1-substituted phenyl-1H-tetrazole-5-carboxylate
ethylesters 4a in moderate yields over two steps (63%–65%). Then compounds 4a reduced by
diisobutylaluminium hydride (DIBAL-H) to form 1-substituted phenyl-1H-tetrazole-5-aldehydes 5a
–f
in good yields (90%–95%). Treatment of 2a
–f with
–
f, which were reacted
–
f
–f
–
f

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in moderate yields (75%–80%). Meanwhile, the side chains 6a and 6b were prepared according the
literature protocol [26]. Subsequently, the compounds 5a were respectively treated with compound
6a and 6b by reductive amination using sodium triacetoxyborohydride (STAB) in dichloroethane to
generate compounds 7a and 8a in moderate yields (75%–85%). Finally, the target compounds 9a
and 10a were obtained by deprotection with saturated hydrochloric acid ethanol solution in good
yields (92%–98%).
–f
–
f
–
e
–f
–e
Scheme 1. Syntheses of 9a
CH2Cl2, RT; ( ) Ph3P, CCl4, reflux; (
DCE, RT; (f) HCl/EtOH, RT.
–
f
and 10a
–
c
e
. Reagents and conditions: (
a
) ethyl oxalyl monochloride, Et3N,
b
) NaN3, CH3CN, RT; ( ) DIBAL-H, CH2Cl2,
d
−
78 ◦C; (
e) STAB,
Scheme 2. Syntheses of 16a
Et3N, ethyl acetate, 0 ◦C; (
) CF3CH2OTf, K2CO3, DMF, RT; (
DCE, RT; (f) HCl/EtOH, RT.
–
e
. Reagents and conditions: (
a
) BBr3, CH2Cl2,
−
78◦C - RT; (
78 ◦C; (
e
b
) CH3SO2Cl,
c
d
) DIBAL-H, CH2Cl2,
−
) STAB,

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Scheme 3. Syntheses of 18a
–e and 20. Reagents and conditions: (a) STAB, DCE, RT; (b) HCl/EtOH, RT;
(c) 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea, Et3N, HgCl2, DMF, RT.
The syntheses of tetrazole derivatives 16a–e were depicted in Scheme 2. The compounds
4c–d could be converted into 11c–d by demethylation using boron tribromide in the yield of
67%. Then the treatment of compounds 11c–d with methanesulfonyl chloride in the presence
of triethylamine by sulfonylation generated 12a
with 2,2,2-trifluoroethyltrifluoromethanesulfonate in the presence of potassium carbonate by alkylation
to give 13a . The intermediate 12a and 13a were directly reduced by DIBAL-Hto form 14a in
moderate yields over two steps (75%–78%). Subsequently similar with the procedure in Scheme 1,
compounds 14a were respectively reacted with side chains 6a and 6b by reductive amination
–b. Meanwhile, the compounds 11c–d were reacted
–b
–b
–b
–d
–
d
to generated compounds 15a–e, which were deprotected with saturated hydrochloric acid ethanol
solution to afford the target compounds 16a–e in good yields (92%–95%).
The syntheses of tetrazole derivatives 18a
were commercially available, and the preparation of the side chains 6f
methods [24 27]. Then the compound 5a was reacted with varied side chains 6c
to generate the compounds 17a in moderate yields (75%–85%). The target compounds 18a–e
–
e
and 20 were illustrated in Scheme 3. The side chains
was followed the reported
by reductive amination
were
6c
–
e
–g
,
–g
–
e
obtained by deprotection with saturated hydrochloric acid ethanol solution in good yields (92%–95%).
Moreover, the compound 18a was reacted with 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea
in the presence of triethylamine and mercury dichloride to give compound 19 in the yield of 71%.

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Finally, the target compound 20 was afforded by deprotection with saturated hydrochloric acid ethanol
solution in the yield of 92%.
2.3. In vitro PRMT1 Inhibition Assays and Selectivity Assays
A series of 1,5-substituded tetrazole derivatives were synthesized to investigate the
structure-activity relationship (SAR) of PRMT1 inhibitors (Table 1). Initially, we designed and
synthesized a series of molecules (9a–e, 16a–d, Group I) as shown in Table 1, which contained
substituted phenyl on 1H-tetrazol and ethylenediamine side chain. The chemical modification of
these compounds is mainly focused on the substituted benzene ring. The initial screening of these
compounds was carried out using the radioactive PRMT1 methylation inhibition assay, which measured
the amount of methyl groups that transferred from [3H]-SAM to a biotinylated histone H4 peptide
(ac- SGRGKGGKGLGKGGAKRHRKVGGK(Biotin)). In the assay, SAH and AMI-1 were used as
the positive controls. It was found that, in Group I, the inhibitory activities of the para-substitution
compounds were in general better than those with meta-substitution. Three compounds (9a
16c), which contained 4-OCH(CH3)2, 4-OCF3 and 4-OCH2CF3 respectively, showed strong inhibitory
effects (over 47%) at 10 M against PRMT1 in the initial screening, and were selected for the IC50
determinations, which were 3.5 M, 23.8 M and 19.9 M, respectively. It should be noted that the
, 9f,
µ
µ
µ
µ
IC50 of compound 9a is around seven times weaker than SAH but around 20 times stronger than
AMI-1 (Table 1). In the second step, the Group II compounds were designed and synthesized by
extending the ethylenediamine side chain to propylenediamine. However, the inhibitory activities of
all the compounds in the Group II (10a
comparison with those of Group I. In the third step, in order to evaluate the influence of other amino
side chains, we designed and synthesized a series of compounds (18a 20, Group III) based on the
structure of compound 9a. Among these compounds, 18a and 18e exhibited 10.0 M and 29.0
IC50s respectively against PRMT1, while the other compounds (18b 20) showed relatively poor
–e, 16e) were either totally abolished or remarkably reduced in
–e,
µ
µM
–d,
activities and their inhibitory effects were all lower than 39% at a concentration of 10 µM.
To further investigate the selectivity profiles of compounds 9a (represented in Figure 4), 9f
,
16c,
18a and 18e, the IC50s against PRMT5 (the type II PRMT) were measured over 100 µM, as illustrated in
Table 1. It suggested that in comparison with type II PRMT, these inhibitors showed good selectivity
against PRMT1.
Figure 4. Inhibitory data of Compound 9a against PRMT1 and PRMT5. The IC50 determination for 9a
against the PRMT1 (A) and PRMT5 (B) based on the radioactive methylation inhibition assay.

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Table 1. Inhibitory activities of 1,5-substituded tetrazole derivatives against PRMT1 and PRMT5.
IC50 (µM)
% Inhibition of PRMT1
Group
Compounds
R1
R2
(at 10 µM) a
PRMT1
PRMT5
9a
9b
9c
4-OCH(CH3)2
3-OCH(CH3)2
4-OCH3
65
0
3.5
>100
-
-
I
3
-
-
9d
9f
3-OCH3
7
-
-
4-OCF3
47
−17
23
13
23.8
>100
9e
3-OCF3
-
-
-
-
-
-
16a
16b
4-OSO2CH3
3-OSO2CH3
16c
4-OCH2CF3
3-OCH2CF3
52
3
19.9
-
>100
16d
-
10a
10b
10c
10d
4-OCH(CH3)2
3-OCH(CH3)2
4-OCH3
23
0
-
-
-
-
-
-
-
-
II
−6
−14
3-OCH3
10e
16e
3-OCF3
−12
−4
-
-
-
-
3-OSO2CH3
18a
18b
4-OCH(CH3)2
4-OCH(CH3)2
50
15
10.0
-
>100
-
III
18c
4-OCH(CH3)2
4-OCH(CH3)2
10
18
-
-
-
-
18d
18e
20
4-OCH(CH3)2
4-OCH(CH3)2
39
29.0
-
>100
−7
-
SAH
AMI-1
0.49
72
0.40
-
a
Initial inhibition rate was tested at 10
selected for IC50 measurements.
µM for all compounds, and only those with >40% inhibition effects were

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2.4. Mechanism of Action (MOA) Study of the Compound 9a
The most potent PRMT1 inhibitor in the current study, compound 9a, was selected for the
Mechanism of Action (MOA) study. As illustrated in the Figure 5, various concentrations of SAM
or peptide were used to evaluate the potency (IC50) of 9a, and no significant changes were observed
according to the concentration changes of either the SAM or peptide. The result of the MOA studies
showed that the compound 9a is a noncompetitive inhibitor for either the cofactor SAM or peptide
substrate. Does this result indicate that the compound 9a inhibit PRMT1 by binding to an allosteric
site? Both PRMT1 and PRMT6 are Type I PRMTs, and their structures share the high degree similarity.
The co-crystal structure of PRMT6 and its inhibitor, MS023, which also contains ethylenediamine
moiety, was solved and clearly showed that ethylenediamine moiety occupied the substrate arginine
binding site. Based on the crystal structure, it can be predicated that MS023 should be a competitive
inhibitor against the peptide. However, similar to our result, the MOA results showed that the
MS023 was noncompetitive with either SAM or peptide substrate [28]. Therefore, we believed that
compound 9a may occupied the substrate arginine binding site, although the MOA results did not
show competitive inhibition effects with respect to the peptide. There are potential explanations for
the current contradictory results. First, the main binding effects of the substrate were from the outside
regions of arginine-binding site. Therefore, compound 9a did occupy the arginine-binding site, but it
did not affect the binding of the whole substrate peptides. Second, the binding of compound 9a
possibly induced major protein conformational changes, which caused the peptides could not enter
the binding site to compete with compound 9a even at the high concentration of the peptides.
Figure 5. Mechanism of Action (MOA) study results. (A,C) Competition assay with SAM on PRMT1.
(B,D) Competition assay with Peptide on PRMT1.
2.5. Molecular Modeling Study of the Interactions between Compound 9a and hPRMT1-SAH Complex
To gain more detailed information regarding the interactions between the compound 9a and
hPRMT1-SAH complex, it was docked into the protein by using the position of substrate arginine
binding site as the reference. There was 100 ns molecular dynamic simulation performed on the complex,
and then the stable trajectory was collected for the binding free energy calculations ( 9.64 kcal/mol)
−
by using MM-PBSA and Normal Mode as shown in Table 2, which indicated a strong binding of
compound 9a. It was noticed that, in the stable MD trajectory, the compound 9a occupied the substrate
arginine binding site (Figure 6A), in which N, N’-dimethylethylenediamine mimicked the guanidyl
group of the arginine substrate and bound closely to the SAH, and the binding pose of compound
9a was stably maintained during the whole simulation process. The free energy decomposition was

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performed and the residues whose binding free energy contributions greater than –0.70 kcal/mol were
recorded (Figure 6C). It was noticed that the Tyr47, Ile52, Met56, Asp84, Glu108, Glu152, Met154, Tyr156
and Tyr160 formed strong interactions with the compound 9a (Figure 6B). Among these residues,
Tyr47, Ile52, Met56 and Tyr156 formed strong van der Walls interactions, in which Tyr47 and Tyr156
formed π-π interactions with the aromatic ring of compound 9a, and three negatively charged residues
(Asp84, Glu108 and Glu152) contributed large electrostatic interactions because of the positive charges
of compound 9a. The MD simulations also showed that the compound 9a could form three stable
hydrogen bonds with Tyr156, Met154 and Glu152 along the stable trajectory. By using the molecular
dynamic simulations, we believed that the binding site of compound 9a is at the substrate arginine
binding position, so that the methylation process could be disturbed. However, the molecular dynamic
simulation results need to be further validated by experimental data, such as the key residues mutations
in the active site or co-crystallization, which will be considered in our following work.
Table 2. Calculated energy components, binding free energy (kcal/mol) of compound 9a in hPRMT1.
∆Gpb
−257.49 268.40
± 0.70 0.49
∆Gmmpbsa
∆Gbinding
−9.64
Simulations
∆Evdw
∆Eele
∆Gnp
∆Ggas
−296.07 263.43
± 0.70 0.49
∆Gsolv
T∆S
−38.58
± 0.22
±
−4.98 ±
±
−32.64 ±
−23.00
± 3.29
9a
0.01
0.49
Figure 6. (A) The binding of SAH and compound 9a (carbon atoms were colored in purple) with
hPRMT1(gray = basic regions, ochre = acidic regions, orange = polar regions, cyan = nonpolar regions);
) The interactions between key residues (carbon atoms were colored in cyan) and compound 9a
(
B
(carbon atoms were colored in green). The hydrogen bonds were represented by black dotted lines;
(C) The residues whose binding free energy contributions were greater than −0.7 Kcal/mol.
2.6. The Alteration of 9a on PRMT1 Patterns at the Cellular Level
Western blotting was performed to confirm that compound 9a altered the arginine methylation
patterns of PRMT1 in the highly metastatic breast cancer cell line MDA-MB-231, as it has been
reported that the expression of PRMT1 is high in the MDA-MB-231 [29]. Before the western blotting
experiment was performed, the potential cytotoxic effect of compound 9a against MDA-MB-231 (breast
cancer) cells was examined using WST-8/CCK8 cell viability assays. The results showed the IC50
of compound 9a against MDA-MB-231 for 48 h was much higher than 500
the time to 72 h, and the IC50 was obtained as 541.1 6.5 M. Therefore, the top concentration of
compound 9a for the western blotting experiment was chosen as 200 M, and during the experiment,
µM. We then extended
±
µ
µ

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no obvious cell damage was observed. As shown in Figure 7, after the treatment of MDA-MB-231
cells with 9a for 48 h, the global level of ADMA, which is mainly produced by the type I PRMTs,
was significantly decreased in a concentration-dependent manner. It was also noticed that the level
of dimethylarginine dimethylaminohydrolases (DDAH), which can metabolize more than 90% of
ADMA [30], was significantly decreased in a concentration-dependent manner. The results showed
that the decreasing of ADMA was because of the inhibition of PRMT1 rather than being metabolized
by DDAH, which indicated that the compound 9a influenced the PRMT-AMDA pathway. Asymmetric
dimethylation of histone H4 at arginine 3 (H4R3me2as) is mediated by PRMT1, and it was observed
that the compound 9a decreased the global level of H4R3me2as in a concentration-dependent manner.
The global level of SDMA, which was catalyzed by Type II PRMTs, was not significantly affected.
By combining the above information, it could be confirmed that 9a has significant influences on PRMT
pathway at cellular levels.
Figure 7. After the treatment of MDA-MB-231 cells with 9a for 48 h, the western blotting results
showed that the global levels of asymmetrical dimethylarginine (ADMA), dimethylaminohydrolases
(DDAH) and H4R3me2a were significantly decreased in concentration-dependent manners, and the
global level of symmetrical dimethylarginine (SDMA) was not significantly changed. On the Wnt
pathway, the western blotting results showed that the global levels of Wnt3A and β-catenin were
significantly decreased in concentration-dependent manners, while the level of Wnt5a/b was not
changed. The experiments were repeated in triplicates.
It was reported that the PRMT1 is required for canonical Wnt signaling [31], and another recent
paper reported that the depletion of PRMT1 blocked Wnt-induced micropinocytosis [32], which
reminded us to see if the inhibition of PRMT1 by compound 9a shows influences on the Wnt signaling
pathways. The global levels of Wnt3a/
β-catenin (Canonical Wnt signaling) and Wnt5a (Non-canonical
Wnt signaling) were evaluated, in which the levels of Wnt3a and
β-catenin were decreased in
concentration-dependent manners, and the level of Wnt5a did not change significantly. The results
suggested that the inhibition of PRMT1 by compound 9a selectively down regulated the canonical
Wnt signaling pathway, which was of potential medical interests. To further confirm the relationships
between PRMT1 inhibitors and Wnt pathway, more factors or biomarkers need to be evaluated in
the future work, such as matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 9 (MMP9),
and methylation of Ras GTPase-activating protein-binding protein 1 (G3BP1) etc.

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3. Materials and Methods
3.1. General Information
All the required chemicals were purchased from commercial sources and used without further
purification. TLC was performed on silica gel 60 pre-coated aluminium plates (0.20 mm thickness)
from Macherey-Nagel and visualisation was accomplished with UV light (254 nm). Compounds were
1
purified by flash column chromatography using 80–100 mesh silica gel. HNMR and 13CNMRspectra
were obtained from Bruker AVANCE III 400 spectrometers using chloroform-d, DMSO-d6 or deuterium
oxide as a solvent. The chemical shifts, given as
δ values, were quoted in parts per million (ppm);
1HNMR chemical shifts were measured relative to internal tetramethylsilane; multiplicities quoted as
singlet (s), doublet (d), triplet (t), quartet (q) or combinations thereof as appropriate. HRMS spectra
were obtained on a Thermo Q-Exactive Orbitrap mass spectrometer. Melting points were determined
on a WRS-1B apparatus without corrected.
3.2. Chemical Synthesis
3.2.1. Synthesis of 9a–f and 10a–e
General procedure for the preparation of compounds 2a–f.
Triethylamine (2.5 eq) was added to the substituted aniline 1a–f (1.0 eq) dissolved in anhydrous
dichloromethane (1.5 mL/mmol). The reaction mixture was cooled to 0 ◦C and ethyl oxalyl monochloride
(1.2 eq) was added dropwise to the solution. Subsequently, the reaction was warmed to room temperature
and stirred for 1 h. The reaction mixture was quenched with water and extracted with dichloromethane,
the organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was
purified by column chromatography on silica gel using EtOAc/petroleum as eluent to give 2a–f.
Ethyl N-(4-isopropoxyphenyl)oxamate (2a)
Yellow solid. Yield: 94%. MP: 128.7–129.9 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 10.67 (s, 1H, -NH-),
7.69–7.55 (m, 2H, -ArH), 6.98–6.77 (m, 2H, -ArH), 4.57 (Hept, J = 6.0 Hz, 1H, -OCH(CH3)2), 4.30 (q, J =
7.1 Hz, 2H, -CH2-), 1.31 (t, J = 7.1 Hz, 3H, -CH3), 1.26 (s, 3H, -CH3), 1.24 (s, 3H, -CH3).
Ethyl N-(3-isopropoxyphenyl)oxamate (2b)
1
Yellow oil. Yield: 92%. H NMR (400 MHz, Chloroform-d)
δ 8.84 (s, 1H, -NH-), 7.39 (t, J = 2.2 Hz,
1H, -ArH), 7.26 (t, J = 8.1 Hz, 1H, -ArH), 7.09 (ddd, J = 8.1, 2.1, 1.0 Hz, 1H, -ArH), 6.74 (ddd, J = 8.3, 2.5,
0.9 Hz, 1H, -ArH), 4.58 (p, J = 6.1 Hz, 1H, -OCH(CH3)2), 4.44 (q, J = 7.1 Hz, 2H, -CH2-), 1.46 (t, J = 7.2
Hz, 3H, -CH3), 1.36 (d, J = 6.1 Hz, 6H, -CH(CH3)2).
Ethyl N-(4-methoxyphenyl)oxamate (2c)
Yellow solid. Yield: 94%. MP: 105.7–106.3 ◦C. 1H NMR (400 MHz, Chloroform-d)
δ 8.81 (s, 1H,
-NH-), 7.61–7.56 (m, 2H, -ArH), 6.95–6.90 (m, 2H, -ArH), 4.44 (q, J = 7.1 Hz, 2H, -CH2-), 3.83 (s, 3H,
-OCH3), 1.45 (t, J = 7.1 Hz, 3H, -CH3).
Ethyl N-(3-methoxyphenyl)oxamate (2d)
Yellow solid. Yield: 95%. MP: 88.9–99.3 ◦C. 1H NMR (400 MHz, Chloroform-d)
δ 8.87 (s, 1H,
-NH-), 7.41 (t, J = 2.3 Hz, 1H, -ArH), 7.30 (d, J = 8.1 Hz, 1H, -ArH), 7.13 (ddd, J = 8.0, 2.0, 0.9 Hz, 1H,
-ArH), 6.77 (ddd, J = 8.3, 2.5, 0.9 Hz, 1H, -ArH), 4.44 (q, J = 7.2 Hz, 2H, -CH2-), 3.84 (s, 3H, -OCH3), 1.46
(t, J = 7.2 Hz, 3H, -OCH3), 1.46 (t, J = 7.2 Hz, 3H, -CH3).
Ethyl N-(3-trifluoromethoxyphenyl)oxamate (2e)
1
Yellow oil. Yield: 90%. H NMR (400 MHz, Chloroform-d)
δ 8.96 (s, 1H, -NH-), 7.70 (d, J = 2.5 Hz,
1H, -ArH), 7.54 (ddd, J = 8.3, 2.1, 0.9 Hz, 1H, -ArH), 7.42 (t, J = 8.2 Hz, 1H, -ArH), 7.08 (ddt, J = 8.3, 2.3,
1.1 Hz, 1H, -ArH), 4.46 (q, J = 7.2 Hz, 2H, -CH2-), 1.46 (t, J = 7.2 Hz, 3H, -CH3).
Ethyl N-(4-trifluoromethoxyphenyl)oxamate (2f)
Yellow solid. Yield: 92%. MP: 131.2–131.9 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 11.01 (s, 1H, -NH-),
7.90–7.85 (m, 2H, -ArH), 7.42–7.36 (m, 2H, -ArH), 4.32 (q, J = 7.1 Hz, 2H, -CH2-), 1.32 (t, J = 7.1 Hz,
3H, -CH3).

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General procedure for the preparation of compounds 3a–f.
2a–f (1.0 eq) was dissolved in CCl4 (2.0 mL/mmol), and a solution of triphenylphosphine in CCl4
(0.8 mL/mmol) was added dropwise to the reaction flask at the room temperature. The reaction was
refluxed and stirred for 6 h, and then cooled to the room temperature. The precipitate was filtered off
and washed with CCl4. The filtrate was concentrated in vacuo without further purification to give 3a
General procedure for the preparation of compounds 4a–f.
–f.
To a solution of 3a–f (1.0 eq) in acetonitrile (1 mL/mmol) was added sodium azide (1.5 eq), and the
reaction was stirred at the room temperature for 3 h and monitored by TLC. The mixture was quenched
with ice water, concentrated and extracted with ethyl acetate. The combined organic layers were
washed with water, and dried over anhydrous Na2SO4, filtered and concentrated. The residue was
purified by column chromatography on silica gel using EtOAc/petroleum as eluent to give 4a–f.
Ethyl 1-(4-isopropoxyphenyl)-1H-tetrazole-5-carboxylate (4a)
White solid. Yield: 65%. MP: 150.2–151.9 ◦C. 1H NMR (400 MHz, Chloroform-d)
δ 7.44–7.35 (m,
2H, -ArH), 7.06–6.99 (m, 2H, -ArH), 4.64 (p, J = 6.1 Hz, 1H, -OCH(CH3)2), 4.46 (q, J = 7.1 Hz, 2H, -CH2-),
1.39 (dd, J = 7.9, 6.6 Hz, 9H, -CH3).
Ethyl 1-(3-isopropoxyphenyl)-1H-tetrazole-5-carboxylate (4b)
1
Yellow oil. Yield: 63%. H NMR (400 MHz, DMSO-d6)
δ 7.48 (t, J = 8.1 Hz, 1H, -ArH), 7.35 (t, J =
2.2 Hz, 1H, -ArH), 7.19 (dddd, J = 11.7, 8.4, 2.2, 0.9 Hz, 2H, -ArH), 4.65 (h, J = 6.0 Hz, 1H, -OCH(CH3)2),
4.33 (t, J = 7.1 Hz, 2H, -CH2-), 1.30 (s, 3H, -CH3), 1.28 (s, 3H, -CH3), 1.20 (t, J = 7.1 Hz, 3H, -CH3).
Ethyl 1-(4-methoxyphenyl)-1H-tetrazole-5-carboxylate (4c)
White solid. Yield: 65%. MP: 75.9–76.3 ◦C. 1H NMR (400 MHz, Chloroform-d)
δ 7.46–7.41 (m, 2H,
-ArH), 7.10–7.05 (m, 2H, -ArH), 4.47 (q, J = 7.1 Hz, 2H, -CH2-), 3.92 (s, 3H, -OCH3), 1.41 (t, J = 7.1 Hz,
3H, -CH3).
Ethyl 1-(3-methoxyphenyl)-1H-tetrazole-5-carboxylate (4d)
White solid. Yield: 64%. MP: 70.1–72.0 ◦C. 1H NMR (400 MHz, Chloroform-d)
δ 7.49 (t, J = 8.1 Hz,
1H, -ArH), 7.16 (ddd, J = 8.5, 2.5, 0.9 Hz, 1H, -ArH), 7.08 (ddd, J = 7.8, 2.0, 0.9 Hz, 1H, -ArH), 7.05 (t, J =
2.2 Hz, 1H, -ArH), 4.48 (q, J = 7.1 Hz, 2H, -CH2-), 3.89 (s, 3H, -OCH3), 1.40 (t, J = 7.1 Hz, 3H, -CH3).
Ethyl 1-(3-trifluoromethoxyphenyl)-1H-tetrazole-5-carboxylate (4e)
Yellow solid. Yield: 64%. MP: 125.2–126.0 ◦C. 1H NMR (400 MHz, Chloroform-d)
δ 7.66 (dd, J =
8.7, 7.7 Hz, 1H, -ArH), 7.51 (dd, J = 7.9, 2.2 Hz, 2H, -ArH), 7.46 (q, J = 1.9 Hz, 1H, -ArH), 4.49 (q, J = 7.1
Hz, 2H, -CH2-), 1.41 (t, J = 7.1 Hz, 3H, -CH3).
Ethyl 1-(4-trifluoromethoxyphenyl)-1H-tetrazole-5-carboxylate (4f)
Yellow solid. Yield: 65%. MP: 68.2–69.0 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 7.96–7.84 (m, 2H,
-ArH), 7.71–7.60 (m, 2H, -ArH), 4.32 (q, J = 7.1 Hz, 2H, -CH2-), 1.19 (t, J = 7.1 Hz, 3H, -CH3).
General procedure for the preparation of compounds 5a–f.
To a solution of 4a
–
f
(1.0 eq) in anhydrous dichloromethane (◦1.8 mL/mmol) was added diisobutyl
aluminium hydride (1.0M in hexanes, 2.0 eq) dropwise at 78 C and the reaction was stirred for
−
30 min. The mixture was quenched with methanol, concentrated and extracted with ethyl acetate.
The combined organic layers were washed with water, 1 M HCl and dried over anhydrous Na2SO4,
filtered and concentrated. The residue was purified by column chromatography on silica gel using
EtOAc/petroleum as eluent to give 5a–f.
1-(4-Isopropoxyphenyl)-1H-tetrazole-5-carbaldehyde (5a)
White solid. Yield: 80%. MP: 122.5–123.1 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 10.05 (s, 1H, -CHO),
7.67–7.63 (m, 2H, -ArH), 7.17–7.13 (m, 2H, -ArH), 4.79–4.72 (m, 1H, -OCH(CH3)2), 1.33 (s, 3H, -CH3),
1.32 (s, 3H, -CH3).
1-(3-Isopropoxyphenyl)-1H-tetrazole-5-carbaldehyde (5b)
1
Colorless oil. Yield: 75%. H NMR (400 MHz, DMSO-d6)
δ 10.07 (s, 1H, -CHO), 7.53 (t, J = 8.1 Hz,
1H, -ArH), 7.38 (t, J = 2.2 Hz, 1H, -ArH), 7.29–7.25 (m, 2H, -ArH), 4.69–4.64 (m, 1H, -OCH(CH3)2), 1.31
(d, J = 0.8 Hz, 3H, -CH3), 1.29 (d, J = 0.8 Hz, 3H, -CH3).
1-(4-Methoxyphenyl)-1H-tetrazole-5-carbaldehyde (5c)

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1
Colorless oil. Yield: 77%. H NMR (400 MHz, Chloroform-d)
2H, -ArH), 7.11–7.07 (m, 2H, -ArH), 3.93 (s, 3H, -OCH3).
δ
10.33 (s, 1H, -CHO), 7.51–7.47 (m,
1-(3-Methoxyphenyl)-1H-tetrazole-5-carbaldehyde (5d)
1
Colorless oil. Yield: 75%. H NMR (400 MHz, DMSO-d6)
δ
10.07 (s, 1H, -CHO) 7.59–7.54 (t, 1H,
-ArH), 7.41 (t, J = 2.2 Hz, 1H, -ArH), 7.31 (dddd, J = 7.9, 6.8, 2.0, 0.9 Hz, 2H, -ArH), 3.83 (s, 3H, -OCH3).
1-(3-Trifluoromethoxyphenyl)-1H-tetrazole-5-carbaldehyde (5e)
Yellow solid. Yield: 78%. MP: 97.6–98.0 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 10.11 (s, 1H, -CHO),
7.96–7.91 (m, 2H, -ArH), 7.75–7.70 (m, 1H, -ArH), 7.65 (ddt, J = 8.0, 2.3, 1.1 Hz, 1H, -ArH).
1-(4-Trifluoromethoxyphenyl)-1H-tetrazole-5-carbaldehyde (5f)
◦
1
Yellow solid. Yield: 80%. MP: 118.7–119.5 C. H NMR (400 MHz, DMSO-d6)
-CHO), 7.94–7.91 (m, 2H, -ArH), 7.70 (dtd, J = 8.1, 2.2, 1.2 Hz, 2H, -ArH).
General procedure for the preparation of compounds 7a–f and 8a–e.
δ 10.10 (s, 1H,
To a solution of 5a–f (1.0 eq) in 1,2-Dichloroethane (8.5 mL/mmol) was added sodium
triacetoxyborohydride (2.0 eq) and 6a or 6b (1.0 eq) and the reaction was stirred for 12 h at room
temperature. The reaction mixture was diluted with water and extracted with dichloromethane.
The dichloromethane layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue
was purified by column chromatography on silica gel using EtOAc/petroleum as eluent to give 7a–f
or 8a–e.
N-tert-Butyloxycarbonyl-N-methyl-N’-(1-(4-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-N’-
methyl-ethylenediamine (7a)
1
Colorless oil. Yield: 85%. H NMR (400 MHz, Chloroform-d)
δ 7.59 (d, J = 8.5 Hz, 2H, -ArH), 7.05
(d, J = 8.9 Hz, 2H, -ArH), 4.65 (p, J = 6.0 Hz, 1H, -OCH(CH3)2), 3.82 (s, 2H, -CH2-), 3.31 (s, 2H, -CH2-),
2.78 (s, 3H, -CH3), 2.38 (s, 3H, -CH3), 1.44 (s, 9H, -C(CH3)3), 1.41 (s, 3H, -CH3), 1.40 (s, 3H, -CH3).
N-tert-Butyloxycarbonyl-N-methyl-N-(1-(3-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-N’-
methyl-ethylenediamine (7b)
1
Colorless oil. Yield: 82%. H NMR (400 MHz, DMSO-d6)
δ 7.52 (t, J = 8.1 Hz, 1H, -ArH), 7.35–7.24
(m, 2H, -ArH), 7.17 (ddd, J = 8.4, 2.5, 0.9 Hz, 1H, -ArH), 4.71 (p, J = 6.0 Hz, 1H, -OCH(CH3)2), 3.11 (t, J =
6.4 Hz, 2H, -CH2-), 2.63 (d, J = 6.4 Hz, 3H, -CH3), 2.48 (t, J = 6.6 Hz, 2H, -CH2-), 2.20 (d, J = 6.4 Hz, 3H,
-CH3), 1.31 (dd, J = 12.4, 8.1 Hz, 15H, -CH3, -C(CH3)3).
N-tert-Butyloxycarbonyl-N-methyl-N’-(1-(4-methoxyphenyl)-1H-tetrazol-5-ylmethyl)-N’-methyl-
ethylenediamine (7c)
1
Colorless oil. Yield: 85%. H NMR (400 MHz, DMSO-d6)
δ 7.66 (dd, J = 8.3, 2.8 Hz, 2H, -ArH),
7.21–7.11 (m, 2H, -ArH), 3.85 (s, 3H, -CH3), 3.83 (s, 2H, -CH2-), 3.15 (d, J = 6.3 Hz, 2H, -CH2-), 2.64 (d, J
= 9.5 Hz, 3H, -CH3), 2.47 (d, J = 6.5 Hz, 2H, -CH2-), 2.18 (d, J = 6.8 Hz, 3H, -CH3), 1.33 (d, J = 12.1 Hz,
9H, -C(CH3)3).
N-tert-Butyloxycarbonyl-N-methyl-N’-(1-(3-methoxyphenyl)-1H-tetrazol-5-yl-methyl)-N’-methyl-
ethylenediamine (7d)
1
Colorless oil. Yield: 83%. H NMR (400 MHz, DMSO-d6)
δ 7.55 (t, J = 8.1 Hz, 1H, -ArH), 7.39–7.30
(m, 2H, -ArH), 7.20 (ddd, J = 8.4, 2.6, 0.9 Hz, 1H, -ArH), 3.90 (s, 2H, -CH2-), 3.84 (s, 3H, -CH3), 3.13 (d, J
= 7.2 Hz, 2H, -CH2-), 2.62 (d, J = 6.6 Hz, 3H, -CH3), 2.48 (d, J = 6.6 Hz, 2H, -CH2-), 2.21 (s, 3H, -CH3),
1.32 (d, J = 10.5 Hz, 9H, -C(CH3)3).
N-tert-Butyloxycarbonyl-N-methyl-N’-(1-(3-trifluoromethoxyphenyl)-1H-tetrazol-5-yl-methyl)-
N’-methyl-ethylenediamine (7e)
1
Colorless oil. Yield: 82%. H NMR (400 MHz, DMSO-d6)
δ
7.93 (s, 1H, -ArH), 7.87–7.82 (m, 1H,
-ArH), 7.79 (t, J = 8.1 Hz, 1H, -ArH), 7.68 (ddt, J = 8.0, 2.4, 1.2 Hz, 1H, -ArH), 3.94 (s, 2H, -CH2-), 3.09 (t, J
= 6.3 Hz, 2H, -CH2-), 2.59 (d, J = 5.4 Hz, 3H, -CH3), 2.46 (t, J = 6.6 Hz, 2H, -CH2-), 2.19 (s, 3H, -CH3),
1.31 (d, J = 9.5 Hz, 9H, -C(CH3)3).
N-tert-Butyloxycarbonyl-N-methyl-N’-(1-(4-trifluoromethoxyphenyl)-1H-tetrazol-5-yl-methyl)-
N’-methyl-ethylenediamine (7f)

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1
Colorless oil. Yield: 84%. H NMR (400 MHz, DMSO-d6)
δ
7.94 (d, J = 8.3 Hz, 2H, -ArH), 7.69–7.64
(m, 2H, -ArH), 3.91 (s, 2H, -CH2-), 3.13 (t, J = 6.4 Hz, 2H, -CH2-), 2.61 (d, J = 6.3 Hz, 3H, -CH3), 2.48 (d,
J = 6.5 Hz, 2H, -CH2-), 2.18 (d, J = 7.0 Hz, 3H, -CH3), 1.32 (d, J = 11.3 Hz, 9H, -C(CH3)3).
N-tert-Butyloxycarbonyl-N-methyl-N’-(1-(4-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-N’-
methyl-1,3-propylenediamine (8a)
1
Colorless oil. Yield: 80%. H NMR (400 MHz, DMSO-d6)
δ 7.61 (dd, J = 9.1, 2.5 Hz, 2H, -ArH),
7.15–7.10 (m, 2H, -ArH), 4.76–4.69 (m, 1H, -OCH(CH3)2), 3.81 (s, 2H, -CH2-), 2.92 (s, 2H, -CH2-), 2.66 (s,
3H, -CH3), 2.29 (t, J = 7.0 Hz, 2H, -CH2-), 2.10 (s, 3H, -CH3), 1.45 (t, J = 7.2 Hz, 2H, -CH2-), 1.34 (d, J =
11.2 Hz, 9H, -C(CH3)3), 1.31 (d, J = 1.5 Hz, 3H, -CH3), 1.30 (s, 3H, -CH3).
N-tert-Butyloxycarbonyl-N-methyl-N’-(1-(3-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-N’-
methyl-1,3-propylenediamine (8b)
1
Colorless oil. Yield: 77%. H NMR (400 MHz, DMSO-d6)
δ 7.51 (t, J = 8.2 Hz, 1H, -ArH), 7.33
(d, J = 9.6 Hz, 1H, -ArH), 7.28–7.22 (m, 1H, -ArH), 7.21–7.14 (m, 1H, -ArH), 4.70 (p, J = 6.0 Hz, 1H,
-OCH(CH3)2), 3.86 (s, 2H, -CH2-), 2.94 (s, 2H, -CH2-), 2.67 (d, J = 4.2 Hz, 3H, -CH3), 2.29 (t, J = 7.0 Hz,
2H, -CH2-), 2.11 (s, 3H, -CH3), 1.46 (t, J = 7.1 Hz, 2H, -CH2-), 1.33 (d, J = 15.1 Hz, 9H, -C(CH3)3), 1.30 (s,
3H, -CH3), 1.28 (s, 3H, -CH3).
N-tert-Butyloxycarbonyl-N-methyl-N’-(1-(4-methoxyphenyl)-1H-tetrazol-5-yl-methyl)-N’-methyl-
1,3-propylenediamine (8c)
1
Colorless oil. Yield: 75%. H NMR (400 MHz, DMSO-d6)
δ 7.67–7.62 (m, 2H, -ArH), 7.18–7.14 (m,
2H, -ArH), 3.85 (d, J = 4.2 Hz, 3H, -CH3), 3.81 (s, 2H, -CH2-), 2.94 (s, 2H, -CH2-), 2.67 (s, 3H, -CH3), 2.28
(t, J = 7.1 Hz, 2H, -CH2-), 2.10 (s, 3H, -CH3), 1.45 (s, 2H, -CH2-), 1.36 (s, 9H, -C(CH3)3).
N-tert-Butyloxycarbonyl-N-methyl-N’-(1-(3-methoxyphenyl)-1H-tetrazol-5-yl-methyl)-N’-methyl-
1,3-propylenediamine (8d)
1
Colorless oil. Yield: 75%. H NMR (400 MHz, DMSO-d6)
δ 7.54 (t, J = 8.2 Hz, 1H, -ArH), 7.39 (t, J
= 2.3 Hz, 1H, -ArH), 7.30 (dddd, J = 6.8, 2.8, 1.9, 0.9 Hz, 1H, -ArH), 7.23–7.17 (m, 1H, -ArH), 3.87 (s, 2H,
-CH2-), 3.83 (s, 3H, -CH3), 2.93 (s, 2H, -CH2-), 2.66 (s, 3H, -CH3), 2.30 (t, J = 7.1 Hz, 2H, -CH2-), 2.12 (s,
3H, -CH3), 1.46 (t, J = 7.2 Hz, 2H, -CH2-), 1.35 (s, 9H, -C(CH3)3).
N-tert-Butyloxycarbonyl-N-methyl-N’-(1-(3-trifluoromethoxyphenyl)-1H-tetrazol-5-yl-methyl)-
N’-methyl-1,3-propylenediamine (8e)
1
Colorless oil. Yield: 75%. H NMR (400 MHz, DMSO-d6)
δ
7.98 (s, 1H, -ArH), 7.83 (ddd, J = 8.0,
4.1, 2.0 Hz, 2H, -ArH), 7.68 (dtq, J = 7.3, 2.5, 1.2 Hz, 1H, -ArH), 3.90 (s, 2H, -CH2-), 2.87 (s, 2H, -CH2-),
2.65 (s, 3H, -CH3), 2.27 (t, J = 7.1 Hz, 2H, -CH2-), 2.08 (s, 3H, -CH3), 1.42 (d, J = 7.4 Hz, 2H, -CH2-), 1.35
(s, 9H, -C(CH3)3).
General procedure for the preparation of compounds 9a–f and 10a–e.
Compounds 7a–f or 8a–e (0.51 mmol) was added to saturated hydrochloric acid ethanol solution
(10 mL) at room temperature and stirred for 30 min. The reaction solution was filtered and washed
with diethylether (20 mL) to give 9a–f or 10a–e.
N,N’-Dimethyl-N’-(1-(4-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)ethylenediamine
hydrochloride (9a)
White solid. Yield: 98%. MP: 187.4–189.4 ◦C. 1H NMR (400 MHz, Deuterium Oxide)
δ 7.49–7.44
(m, 2H, -ArH), 7.19–7.13 (m, 2H, -ArH), 4.74 (p, J = 5.5, 4.9 Hz, 1H, -OCH(CH3)2), 4.35 (s, 2H, -CH2-),
3.21 (dd, J = 14.9, 6.8 Hz, 2H, -CH2-), 3.15–3.01 (m, 2H, -CH2-), 2.64 (dd, J = 4.9, 2.1 Hz, 3H, -CH3), 2.48
(d, J = 37.8 Hz, 3H, -CH3), 1.31 (s, 3H, -CH3), 1.30 (s, 3H, -CH3). 13C NMR (101 MHz, Deuterium Oxide)
δ
159.2, 150.1, 126.7, 124.9, 117.1, 71.9, 51.8, 49.1, 44.2, 41.3, 33.0, 20.9. HRMS (ESI) m/z [M+H]+ Calcd
for C15H25N6O+: 305.20844. Found: 305.20831.
N,N’-Dimethyl-N’-(1-(3-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)ethylenediamine
hydrochloride (9b)
1
Colorless oil. Yield: 95%. H NMR (400 MHz, Deuterium Oxide)
δ
7.49 (ddd, J = 8.4, 7.4, 0.9 Hz,
1H, -ArH), 7.18 (ddd, J = 8.5, 2.3, 1.1 Hz, 1H, -ArH), 7.09–7.04 (m, 2H, -ArH), 4.63 (p, J = 6.1 Hz, 1H,
-OCH(CH3)2), 4.37 (s, 2H, -CH2-), 3.22 (td, J = 6.2, 1.3 Hz, 2H, -CH2-), 3.13 (td, J = 6.4, 1.3 Hz, 2H,

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-CH2-), 2.61 (s, 3H, -CH3), 2.52 (s, 3H, -CH3), 1.24 (s, 3H, -CH3), 1.22 (s, 3H, -CH3). 13C NMR (101 MHz,
Deuterium Oxide)
δ 158.1, 133.1, 131.3, 119.1, 117.3, 112.8, 72.1, 51.8, 49.2, 44.2, 41.3, 33.0, 20.9. HRMS
(ESI) m/z [M+H]+ Calcd for C15H25N6O+: 305.20844. Found: 305.20828.
N,N’-Dimethyl-N’-(1-(4-methoxyphenyl)-1H-tetrazol-5-yl-methyl)ethylenediamine
hydrochloride (9c)
White solid. Yield: 95%. MP: 163.2–163.6 ◦C. 1H NMR (400 MHz, Deuterium Oxide)
δ 7.45–7.40
(m, 2H, -ArH), 7.14–7.10 (m, 2H, -ArH), 4.51 (s, 2H, -CH2-), 3.81 (s, 3H, -CH3), 3.31 (s, 4H, -CH2-), 2.67
(s, 3H, -CH3), 2.64 (d, J = 0.7 Hz, 3H, -CH3). 13C NMR (101 MHz, Deuterium Oxide)
δ 161.1, 149.8,
126.6, 124.9, 115.3, 55.7, 51.8, 49.1, 44.00, 41.4, 33.0. HRMS (ESI) m/z [M+H]+ Calcd for C13H21N6O+:
277.17714. Found: 277.17715.
N,N’-Dimethyl-N’-(1-(3-methoxyphenyl)-1H-tetrazol-5-yl-methyl)ethylenediamine
hydrochloride (9d)
White solid. Yield: 98%. MP: 185.6–186.4 ◦C. 1H NMR (400 MHz, Deuterium Oxide)
δ 7.52–7.47
(m, 1H, -ArH), 7.18 (ddd, J = 8.5, 2.5, 0.9 Hz, 1H, -ArH), 7.10–7.05 (m, 2H, -ArH), 4.47 (s, 2H, -CH2-),
3.77 (s, 3H, -CH3), 3.29–3.20 (m, 4H, -CH2-), 2.62 (s, 3H, -CH3), 2.60 (s, 3H, -CH3). 13C NMR (101 MHz,
Deuterium Oxide)
δ 160.1, 149.7, 133.0, 131.2, 117.2, 117.1, 110.9, 55.7, 51.9, 49.2, 44.1, 41.4, 33.0. HRMS
(ESI) m/z [M+H]+ Calcd for C13H21N6O+: 277.17714. Found: 277.17703.
N,N’-Dimethyl-N’-(1-(3-trifluoromethoxyphenyl)-1H-tetrazol-5-yl-methyl)ethylenediamine
hydrochloride (9e)
White solid. Yield: 97%. MP: 185.6–186.4 ◦C. 1H NMR (400 MHz, Deuterium Oxide)
δ 7.66 (td,
J = 8.1, 1.5 Hz, 1H, -ArH), 7.58–7.52 (m, 2H, -ArH), 7.52–7.48 (m, 1H, -ArH), 4.49 (s, 2H, -CH2-), 3.28 (d,
J = 4.2 Hz, 2H, -CH2-), 3.26 (d, J = 3.9 Hz, 2H, -CH2-), 2.63 (s, 3H, -CH3), 2.62 (s, 3H, -CH3). 13C NMR
(101 MHz, Deuterium Oxide) δ 150.7, 149.4, 133.2, 131.8, 123.9, 123.5, 118.1, 51.9, 49.4, 44.5, 41.3, 32.9.
HRMS (ESI) m/z [M+H]+ Calcd for C13H18F3N6O+: 331.14887. Found: 331.14871.
N,N’-Dimethyl-N’-(1-(4-trifluoromethoxyphenyl)-1H-tetrazol-5-yl-methyl)ethylenediamine
hydrochloride (9f)
White solid. Yield: 98%. MP: 194.3–195.2 ◦C. 1H NMR (400 MHz, Deuterium Oxide)
δ
7.61–7.56
(m, 2H, -ArH), 7.51–7.46 (m, 2H, -ArH), 4.25 (s, 2H, -CH2-), 3.15 (t, J = 6.4 Hz, 2H, -CH2-), 3.02 (t, J = 6.4
Hz, 2H, -CH2-), 2.58 (s, 3H, -CH3), 2.42 (s, 3H, -CH3). 13C NMR (101 MHz, Deuterium Oxide)
δ 150.6,
150.3, 130.6, 126.9, 122.5, 51.9, 49.3, 44.3, 41.3, 33.0. HRMS (ESI) m/z [M+H]+ Calcd for C13H18F3N6O+:
331.14887. Found: 331.14871.
N,N’-Dimethyl-N’-(1-(4-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-1,3-propylenediamine
hydrochloride (10a)
Colorless oil. Yield: 94%. 1H NMR (400 MHz, Deuterium Oxide)
δ 7.43–7.39 (m, 2H, -ArH),
7.14–7.09 (m, 2H, -ArH), 4.76 (d, J = 1.2 Hz, 2H, -CH2-), 4.67 (d, J = 6.1 Hz, 1H, -OCH(CH3)2), 3.36–3.27
(m, 2H, -CH2-), 3.05–2.97 (m, 2H, -CH2-), 2.94–2.89 (m, 3H, -CH3), 2.61 (s, 3H, -CH3), 2.14–2.03 (m,
2H, -CH2-), 1.26 (s, 3H, -CH3), 1.25 (s, 3H, -CH3). 13C NMR (101 MHz, Deuterium Oxide)
δ 159.4,
148.0, 126.7, 124.5, 117.2, 71.9, 53.5, 48.1, 45.4, 41.1, 32.71, 20.9. HRMS (ESI) m/z [M+H]+ Calcd for
C16H27N6O+: 319.22409. Found: 319.22406.
N,N’-Dimethyl-N’-(1-(3-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-1,3-propylenediamine
hydrochloride (10b)
1
Colorless oil. Yield: 92%. H NMR (400 MHz, Deuterium Oxide)
δ
7.50 (t, J = 8.1 Hz, 1H, -ArH),
7.20 (ddd, J = 8.5, 2.4, 0.9 Hz, 1H, -ArH), 7.09–7.03 (m, 2H, -ArH), 4.79 (s, 2H, -CH2-), 4.68–4.60 (m, 1H,
-OCH(CH3)2), 3.36–3.28 (m, 2H, -CH2-), 3.04–2.96 (m, 2H, -CH2-), 2.91 (s, 3H, -CH3), 2.61 (s, 3H, -CH3),
2.15–2.04 (m, 2H, -CH2-), 1.24 (s, 3H, -CH3), 1.23 (s, 3H, -CH3). 13C NMR (101 MHz, Deuterium Oxide)
δ
158.2, 147.7, 132.7, 131.4, 119.3, 117.3, 112.8, 72.1, 53.6, 48.2, 45.4, 41.1, 32.7, 20.9, 20.8. HRMS (ESI) m/z
[M+H]+ Calcd for C16H27N6O+: 319.22409. Found: 319.22405.
N,N’-Dimethyl-N’-(1-(4-methoxyphenyl)-1H-tetrazol-5-yl-methyl)-1,3-propylenediamine
hydrochloride (10c)

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White solid. Yield: 94%. MP: 188.2–190.1 ◦C. 1H NMR (400 MHz, Deuterium Oxide)
δ
7.48–7.42
(m, 2H, -ArH), 7.19–7.12 (m, 2H, -ArH), 4.73 (s, 2H, -CH2-), 3.84 (s, 3H, -CH3), 3.32–3.24 (m, 2H, -CH2-),
3.05–2.98 (m, 2H, -CH2-), 2.88 (s, 3H, -CH3), 2.64 (s, 3H, -CH3), 2.14–2.04 (m, 2H, -CH2-). 13C NMR (101
MHz, Deuterium Oxide) δ 148.1, 126.7, 124.5, 115.4, 55.7, 53.5, 48.1, 45.4, 41.1, 32.7, 20.9. HRMS (ESI)
m/z [M+H]+ Calcd for C14H23N6O+: 291.19279. Found: 291.19271.
N,N’-Dimethyl-N’-(1-(3-methoxyphenyl)-1H-tetrazol-5-yl-methyl)-1,3-propylenediamine
hydrochloride (10d)
1
Colorless oil. Yield: 92%. H NMR (400 MHz, Deuterium Oxide)
δ
7.51 (dd, J = 8.5, 7.8 Hz, 1H,
-ArH), 7.20 (ddd, J = 8.6, 2.5, 0.9 Hz, 1H, -ArH), 7.09 (t, J = 2.2 Hz, 1H, -ArH), 7.06 (ddd, J = 7.8, 2.1, 0.9
Hz, 1H, -ArH), 4.80 (d, J = 1.3 Hz, 2H, -CH2-), 3.78 (s, 3H, -CH3), 3.36–3.29 (m, 2H, -CH2-), 3.05–2.97 (m,
2H, -CH2-), 2.91 (d, J = 1.3 Hz, 3H, -CH3), 2.61 (s, 3H, -CH3), 2.17–2.04 (m, 2H, -CH2-). 13C NMR (101
MHz, Deuterium Oxide) δ 160.1, 147.8, 132.7, 131.4, 117.4, 117.2, 110.9, 55.8, 53.6, 48.2, 45.4, 41.1, 32.7,
20.8. HRMS (ESI) m/z [M+H]+ Calcd for C14H23N6O+: 291.19279. Found: 291.19267.
N,N’-Dimethyl-N’-(1-(3-trifluoromethoxyphenyl)-1H-tetrazol-5-yl-methyl)-1,3-propylenediamine
hydrochloride (10e)
Colorless oil. Yield: 92%. 1H NMR (400 MHz, Deuterium Oxide)
δ 7.70–7.64 (m, 1H, -ArH),
7.58–7.52 (m, 2H, -ArH), 7.48 (ddd, J = 7.9, 2.0, 1.0 Hz, 1H, -ArH), 4.71 (s, 2H, -CH2-), 3.26–3.20 (m, 2H,
-CH2-), 3.01–2.95 (m, 2H, -CH2-), 2.83 (s, 3H, -CH3), 2.60 (s, 3H, -CH3), 2.10–2.01 (m, 2H, -CH2-). 13C
NMR (101 MHz, Deuterium Oxide) δ 148.2, 132.8, 132.0, 124.2, 123.5, 118.1, 53.7, 48.5, 45.5, 41.1, 32.7,
20.9. HRMS (ESI) m/z [M+H]+ Calcd for C14H20F3N6O+: 345.16452. Found: 345.16431.
3.2.2. Synthesis of 16a–e
General procedure for the preparation of compounds 11c–d.
To a solution of compound 4c
(16.12 mL, 1 M solution in CH2Cl2, 16.12 mmol) dropwise under argon at
mixture was warmed to room temperature and stirred for 12h. The mixture was washed three times
with water, the organic layer was dried over Na2SO4, and the solvent removed under reduced pressure.
The solid residue was purified by column chromatography on silica gel using EtOAc/petroleum as
eluent to give 11c–d.
–
d
(2.00 g, 8.06 mmol) in dry dichloromethane was added BBr3
78 ◦C and then the reaction
−
Ethyl 1-(4-hydroxylphenyl)-1H-tetrazole-5-carboxylate (11c)
White solid. Yield: 67%. MP: 136.4–137.0 ◦C. 1H NMR (400 MHz, Chloroform-d)
δ 7.43–7.37 (m,
2H, -ArH), 7.06–7.00 (m, 2H, -ArH), 4.47 (q, J = 7.1 Hz, 2H, -CH2-), 1.41 (t, J = 7.2 Hz, 3H, -CH3).
Ethyl 1-(3-hydroxylphenyl)-1H-tetrazole-5-carboxylate (11d)
White solid. Yield: 67%. MP: 160.4–161.3 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 10.12 (s, 1H, -OH),
7.39 (t, J = 8.0 Hz, 1H, -ArH), 7.09 (ddd, J = 7.9, 2.1, 1.0 Hz, 1H, -ArH), 7.06 (t, J = 2.1 Hz, 1H, -ArH), 7.02
(ddd, J = 8.2, 2.4, 1.0 Hz, 1H, -ArH), 4.33 (q, J = 7.1 Hz, 2H, -CH2-), 1.21 (t, J = 7.1 Hz, 3H, -CH3).
General procedure for the preparation of compounds 12a–b.
To a solution of 11c–d (0.60 g, 2.56 mmol) in ethyl acetate was added triethylamine (0.71 mL, 5.12
mmol), methanesulfonyl chloride (0.26 mL, 3.33mmol) at 0 ◦C and stirred for 10 min. The reaction
mixture was quenched with water and extracted with ethyl acetate. The organic layer was dried over
Na2SO4, and the solvent removed under reduced pressure without further purification to give 12a
Ethyl 1-(4-methylsulfonylphenyl)-1H-tetrazole-5-carboxylate (12a)
–b.
White solid, Yield: 95%. MP: 135.7–137.8 ◦C. 1H NMR (400 MHz, Chloroform-d)
δ 7.65–7.61 (m,
2H, -ArH), 7.57–7.51 (m, 2H, -ArH), 4.49 (q, J = 7.2 Hz, 2H, -CH2-), 3.28 (s, 3H, -SO3CH3), 1.42 (t, J = 7.1
Hz, 3H, -CH3).
Ethyl 1-(3-methylsulfonylphenyl)-1H-tetrazole-5-carboxylate (12b)
1
Yellow oil, Yield: 95%. H NMR (400 MHz, DMSO-d6)
δ
7.91–7.87 (m, 1H, -ArH), 7.79–7.72 (m, 2H,
-ArH), 7.66 (dt, J = 6.9, 2.3 Hz, 1H, -ArH), 4.32 (q, J = 7.1 Hz, 2H, -CH2-), 3.47 (s, 3H, -SO3CH3), 1.20 (t, J
= 7.1 Hz, 3H, -CH3).
General procedure for the preparation of compounds 13a–b.

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To a solution of 11c
–
d
(0.40 g, 1.71 mmol) in dry DMF was added K2CO3 (0.28 g, 2.05 mmol),
2,2,2-trifluoroethyltrifluoromethanesulfonate (0.27 mL, 1.88 mmol) at room temperature and stirred for
3h. The mixture was quenched with water and extracted with ethyl acetate. The organic layer was
dried over Na2SO4, and the solvent removed under reduced pressure without further purification to
give 13a–b.
Ethyl 1-(4-(2,2,2-trifluoroethoxy)phenyl)-1H-tetrazole-5-carboxylate (13a)
White solid, Yield: 94%. MP: 119.2–120.0 ◦C. 1H NMR (400 MHz, Chloroform-d)
δ 7.53–7.48 (m,
2H, -ArH), 7.16–7.12 (m, 2H, -ArH), 4.47 (qd, J = 7.5, 4.4 Hz, 4H, -CH2CF3, -CH2-), 1.42 (t, J = 7.2 Hz,
3H, -CH3).
Ethyl 1-(3-(2,2,2-trifluoroethoxy)phenyl)-1H-tetrazole-5-carboxylate (13b)
White solid, Yield: 94%. MP: 77.0–77.6 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 7.59 (t, J = 8.2 Hz, 1H,
-ArH), 7.53 (t, J = 2.2 Hz, 1H, -ArH), 7.38 (dddd, J = 9.3, 8.4, 2.3, 1.0 Hz, 2H, -ArH), 4.86 (q, J = 8.8 Hz,
2H, -CH2CF3), 4.33 (q, J = 7.1 Hz, 2H, -CH2-), 1.20 (t, J = 7.1 Hz, 3H, -CH3).
General procedure for the preparation of compounds 14a–d.
To a solution of 12a
diisobutyl aluminium hydride (1.0 M in hexanes, 2.0 eq) dropwise at
stirred for 30 min. The mixture was quenched with methanol, concentrated and extracted with ethyl
acetate. The combined organic layers were washed with water, 1 M HCl and dried over anhydrous
Na2SO4, filtered and concentrated. The residue was purified by column chromatography on silica gel
using EtOAc/petroleum as eluent to give 14a–d.
–
b
or 13a
–
b
(1.0 eq) in anhydrous dichloromethane (1.8 mL/mmol) was added
78 ◦C and the reaction was
−
1-(4-Methylsulfonylphenyl)-1H-tetrazole-5-carbaldehyde (14a)
◦
1
Yellow solid. Yield: 78%. MP: 146.7–147.0 C. H NMR (400 MHz, DMSO-d6)
-CHO), 7.94–7.88 (m, 2H, -ArH), 7.68–7.63 (m, 2H, -ArH), 3.52 (s, 3H, -SO3CH3).
δ 10.10 (s, 1H,
1-(3-Methylsulfonylphenyl)-1H-tetrazole-5-carbaldehyde (14b)
1
Yellow oil. Yield: 75%. H NMR (400 MHz, DMSO-d6)
δ
10.09 (s, 1H, -CHO), 7.87 (t, J = 2.1 Hz,
1H, -ArH), 7.77–7.72 (m, 2H, -ArH), 7.61 (ddd, J = 8.0, 2.3, 1.2 Hz, 1H, -ArH), 3.49 (s, 3H, -SO3CH3).
1-(4-(2,2,2-Trifluoroethoxy)phenyl)-1H-tetrazole-5-carbaldehyde (14c)
1
Yellow oil. Yield: 76%. H NMR (400 MHz, DMSO-d6)
δ
10.06 (s, 1H, -CHO), 7.76–7.72 (m, 2H,
-ArH), 7.33–7.30 (m, 2H, -ArH), 4.92 (q, J = 8.9, 7.1 Hz, 2H, -CH2CF3).
1-(3-(2,2,2-Trifluoroethoxy)phenyl)-1H-tetrazole-5-carbaldehyde (14d)
White solid. Yield: 75%. MP: 101.5–102.0 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ
10.08 (s, 1H, -CHO),
7.63 (t, J = 8.2 Hz, 1H, -ArH), 7.49 (t, J = 2.3 Hz, 1H, -ArH), 7.42 (ddd, J = 5.5, 2.0, 0.9 Hz, 1H, -ArH),
7.41–7.39 (m, 1H, -ArH), 4.87 (q, J = 8.8, 1.3 Hz, 2H, -CH2CF3).
General procedure for the preparation of compounds 15a–e.
To a solution of 14a–d (1.0 eq) in 1,2-Dichloroethane (8.5 mL/mmol) was added sodium
triacetoxyborohydride (2.0 eq) and 6a or 6b (1.0 eq) and the reaction was stirred for 12 h at room
temperature. The reaction mixture was diluted with water and extracted with dichloromethane.
The dichloromethane layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue
was purified by column chromatography on silica gel using EtOAc/petroleum as eluent to give 15a–e.
N-tert-Butyloxycarbonyl-N-methyl-N’-(1-(4-methylsulfonylphenyl)-1H-tetrazol-5-yl-methyl)-N’-
methyl-ethylenediamine (15a)
1
Colorless oil. Yield: 77%. H NMR (400 MHz, DMSO-d6)
δ
7.91 (d, J = 8.6 Hz, 2H, -ArH), 7.65–7.61
(m, 2H, -ArH), 3.91 (s, 2H, -CH2-), 3.50 (s, 3H, -CH3), 3.13 (t, J = 6.5 Hz, 2H, -CH2-), 2.62 (d, J = 15.3
Hz, 3H, -CH3), 2.48 (d, J = 6.5 Hz, 2H, -CH2-), 2.19 (d, J = 6.5 Hz, 3H, -CH3), 1.33 (d, J = 11.8 Hz, 9H,
-C(CH3)3).
N-tert-Butyloxycarbonyl-N-methyl-N’-(1-(3-methylsulfonylphenyl)-1H-tetrazol-5-yl-methyl)-N’-
methyl-ethylenediamine (15b)
1
Colorless oil. Yield: 75%. H NMR (400 MHz, DMSO-d6)
δ 7.84 (d, J = 23.0 Hz, 2H, -ArH), 7.77 (t,
J = 8.1 Hz, 1H, -ArH), 7.64 (ddd, J = 8.1, 2.4, 1.2 Hz, 1H, -ArH), 3.94 (s, 2H, -CH2-), 3.49 (s, 3H, -CH3),

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3.12 (t, J = 6.5 Hz, 2H, -CH2-), 2.62 (d, J = 11.0 Hz, 3H, -CH3), 2.48 (t, J = 6.5 Hz, 2H, -CH2-), 2.20 (d, J =
4.4 Hz, 3H, -CH3), 1.32 (d, J = 8.5 Hz, 9H, -C(CH3)3).
N-tert-Butyloxycarbonyl-N-methyl-N’-(1-(4-(2,2,2-trifluoroethoxy)phenyl)-1H-tetrazol-5-yl-
methyl)-N’-methyl-ethylenediamine (15c)
1
Colorless oil. Yield: 82%. H NMR (400 MHz, DMSO-d6)
δ 7.92–7.86 (m, 1H, -ArH), 7.77–7.66 (m,
2H, -ArH), 7.37–7.33 (m, 1H, -ArH), 4.91 (q, J = 8.8 Hz, 2H, -CH2-), 3.85 (s, 2H, -CH2-), 3.14 (t, J = 6.5 Hz,
2H, -CH2-), 2.64 (d, J = 13.8 Hz, 3H, -CH3), 2.48 (t, J = 6.4 Hz, 2H, -CH2-), 2.18 (s, 3H, -CH3), 1.33 (d,
J = 12.7 Hz, 9H, -C(CH3)3).
N-tert-Butyloxycarbonyl-N-methyl-N’-(1-(3-(2,2,2-trifluoroethoxy)phenyl)-1H-tetrazol-5-yl-
methyl)-N’-methyl-ethylenediamine (15d)
1
Colorless oil. Yield: 80%. H NMR (400 MHz, DMSO-d6)
δ 7.61 (t, J = 8.2 Hz, 1H, -ArH), 7.55–7.40
(m, 2H, -ArH), 7.34 (dd, J = 8.5, 2.5 Hz, 1H, -ArH), 4.89 (q, J = 8.8 Hz, 2H, -CH2-), 3.93 (s, 2H, -CH2-),
3.11 (t, J = 6.5 Hz, 2H, -CH2-), 2.61 (d, J = 12.7 Hz, 3H, -CH3), 2.47 (t, J = 6.5 Hz, 2H, -CH2-), 2.19 (s, 3H,
-CH3), 1.32 (d, J = 9.0 Hz, 9H, -C(CH3)3).
N-tert-Butyloxycarbonyl-N-methyl-N’-(1-(3-methylsulfonylphenyl)-1H-tetrazol-5-yl-methyl)-N’-
methyl-1,3-propylenediamine (15e)
Colorless oil. Yield: 75%.1H NMR (400 MHz, DMSO-d6)
δ 7.90 (t, J = 2.1 Hz, 1H, -ArH), 7.82–7.74
(m, 2H, -ArH), 7.66–7.62 (m, 1H, -ArH), 3.89 (s, 2H, -CH2-), 3.48 (s, 3H, -CH3), 2.93 (s, 2H, -CH2-), 2.67
(d, J = 4.1 Hz, 3H, -CH3), 2.29 (t, J = 7.1 Hz, 2H, -CH2-), 2.10 (s, 3H, -CH3), 1.46 (s, 2H, -CH2-), 1.35 (s,
9H, -C(CH3)3).
General procedure for the preparation of compounds 16a–e.
Compounds 15a–e (0.51 mmol) was added to saturated hydrochloric acid ethanol solution (10 mL)
at room temperature and stirred for 30 min. The reaction solution was filtered and washed with
diethylether (20 mL) to give 16a–e.
N,N’-Dimethyl-N’-(1-(4-methylsulfonylphenyl)-1H-tetrazol-5-yl-methyl)ethylenediamine
hydrochloride (16a)
White solid. Yield: 95%. MP: 198.9–199.3 ◦C. 1H NMR (400 MHz, Deuterium Oxide)
δ 7.65–7.60
(m, 2H, -ArH), 7.56–7.52 (m, 2H, -ArH), 4.24 (s, 2H, -CH2-), 3.32 (s, 3H, -SO3CH3), 3.14 (t, J = 6.4 Hz,
2H, -CH2-), 3.00 (t, J = 6.3 Hz, 2H, -CH2-), 2.58 (s, 3H, -CH3), 2.41 (s, 3H, -CH3). 13C NMR (101 MHz,
Deuterium Oxide) δ 150.2, 150.1, 131.3, 127.1, 124.2, 51.9, 49.4, 44.1, 41.4, 37.1, 33.0. HRMS (ESI) m/z
[M+H]+ Calcd for C13H21N6O3S+: 341.13904. Found: 341.13906.
N,N’-Dimethyl-N’-(1-(3-methylsulfonylphenyl)-1H-tetrazol-5-yl-methyl)ethylenediamine
hydrochloride (16b)
1
Colorless oil. Yield: 94%. H NMR (400 MHz, Deuterium Oxide)
δ
7.68 (td, J = 8.1, 0.7 Hz, 1H,
-ArH), 7.59 (qd, J = 2.5, 0.9 Hz, 2H, -ArH), 7.57–7.53 (m, 1H, -ArH), 4.32 (s, 2H, -CH2-), 3.30 (s, 3H,
-SO3CH3), 3.20–3.15 (m, 2H, -CH2-), 3.06 (t, J = 6.3 Hz, 2H, -CH2-), 2.59 (s, 3H, -CH3), 2.47 (s, 3H, -CH3).
13C NMR (101 MHz, Deuterium Oxide)
δ 150.6, 148.9, 133.3, 132.0, 125.2, 124.3, 119.5, 51.9, 49.3, 44.4,
41.3, 37.1, 32.9. HRMS (ESI) m/z [M+H]+ Calcd for C13H21N6O3S+: 341.13904. Found: 341.13892.
N,N’-Dimethyl-N’-(1-(4-(2,2,2-trifluoroethoxy)phenyl)-1H-tetrazol-5-yl-methyl)ethylenediamine
hydrochloride (16c)
White solid. Yield: 95%. MP: 188.7–190.0 ◦C. 1H NMR (400 MHz, Deuterium Oxide)
δ 7.49–7.44
(m, 2H, -ArH), 7.21–7.15 (m, 2H, -ArH), 4.59 (q, J = 8.4 Hz, 2H, -CH2CF3), 4.25 (s, 2H, -CH2-), 3.16 (t, J =
6.4 Hz, 2H, -CH2-), 3.02 (t, J = 6.4 Hz, 2H, -CH2-), 2.58 (s, 3H, -CH3), 2.43 (s, 3H, -CH3). 13C NMR (101
MHz, Deuterium Oxide)
δ 158.9, 150.1, 126.8, 126.3, 116.3, 65.6, 65.2, 51.8, 49.1, 44.2, 41.3, 33.0. HRMS
(ESI) m/z [M+H]+ Calcd for C14H20F3N6O+: 345.16452. Found: 345.16437.
N,N’-Dimethyl-N’-(1-(3-(2,2,2-trifluoroethoxy)phenyl)-1H-tetrazol-5-ylmethyl)ethylenediamine
hydrochloride (16d)
1
Colorless oil. Yield: 92%. H NMR (400 MHz, Deuterium Oxide)
δ
7.54 (ddd, J = 8.4, 7.6, 0.7 Hz,
1H, -ArH), 7.25 (ddd, J = 8.6, 2.4, 1.0 Hz, 1H, -ArH), 7.20–7.15 (m, 2H, -ArH), 4.57 (q, J = 8.4 Hz, 2H,
-CH2CF3), 4.40 (s, 2H, -CH2-), 3.23 (td, J = 6.2, 1.4 Hz, 2H, -CH2-), 3.16 (dd, J = 7.2, 5.0 Hz, 2H, -CH2-),

Int. J. Mol. Sci. 2019, 20, 3840
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2.61 (s, 3H, -CH3), 2.54 (s, 3H, -CH3). 13C NMR (101 MHz, Deuterium Oxide)
δ
157.9, 131.5, 118.7, 117.6,
112.1, 65.7, 65.3, 51.9, 49.3, 41.2, 32.9. HRMS (ESI) m/z [M+H]+ Calcd for C14H20F3N6O+: 345.16452.
Found: 345.16437.
N,N’-Dimethyl-N’-(1-(3-methylsulfonylphenyl)-1H-tetrazol-5-yl-methyl)-1,3-propylenediamine
hydrochloride (16e)
1
Colorless oil. Yield: 92%. H NMR (400 MHz, Deuterium Oxide)
δ 7.74–7.67 (m, 1H, -ArH), 7.60
(dt, J = 7.7, 1.4 Hz, 2H, -ArH), 7.57–7.52 (m, 1H, -ArH), 4.80 (s, 2H, -CH2-), 3.31 (s, 5H, -CH2-, -SO3CH3),
3.02–2.96 (m, 2H, -CH2-), 2.91 (s, 3H, -CH3), 2.60 (s, 3H, -CH3), 2.13–2.04 (m, 2H, -CH2-). 13C NMR (101
MHz, Deuterium Oxide) δ 149.0, 148.0, 132.8, 132.2, 125.5, 124.3, 119.5, 53.7, 48.4, 45.4, 41.2, 37.1, 32.7,
20.8. HRMS (ESI) m/z [M+H]+ Calcd for C14H23N6O3S+: 355.15469. Found: 355.15460.
3.2.3. Synthesis of 18a–e and 20
General procedure for the preparation of compounds 17a–e.
To a solution of 5a (1.0 eq) in 1,2-Dichloroethane (8.5 mL/mmol) was added sodium
triacetoxyborohydride (2.0 eq) and 6c–g (1.0 eq) and the reaction was stirred for 12 h at room
temperature. The reaction mixture was diluted with water and extracted with dichloromethane. The
dichloromethane layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was
purified by column chromatography on silica gel using EtOAc/petroleum as eluent to give 17a–e.
N-tert-Butyloxycarbonyl-N’-(1-(4-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-N’-methyl-
ethylenediamine (17a)
1
Colorless oil. Yield: 85%. H NMR (400 MHz, Chloroform-d)
δ 7.57–7.43 (m, 2H, -ArH), 7.12–7.03
(m, 2H, -ArH), 4.70–4.61 (m, 1H, -OCH(CH3)2), 3.31 (s, 2H, -CH2-), 2.52 (s, 2H, -CH2-), 1.61 (s, 2H,
-CH2-), 1.45 (s, 9H, -C(CH3)3), 1.42 (d, J = 0.6 Hz, 3H, -CH3), 1.42–1.39 (m, 3H, -CH3), 1.27 (s, 3H, -CH3).
N-tert-Butyloxycarbonyl-N’-(1-(4-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-N’-methyl-1,3-
propylenediamine (17b)
1
Colorless oil. Yield: 80%. H NMR (400 MHz, DMSO-d6)
δ 7.65–7.59 (m, 2H, -ArH), 7.15–7.09 (m,
2H, -ArH), 4.77–4.69 (m, 1H, -OCH(CH3)2), 3.78 (s, 2H, -CH2-), 2.77 (q, J = 6.6 Hz, 2H, -CH2-), 2.32 (t, J =
7.1 Hz, 2H, -CH2-), 2.09 (s, 3H, -CH3), 1.40 (t, J = 6.9 Hz, 2H, -CH2-), 1.36 (s, 9H, -C(CH3)3), 1.32 (s, 3H,
-CH3), 1.31 (d, J = 2.3 Hz, 3H, -CH3).
N-tert-Butyloxycarbonyl-N-methyl-N’-(1-(4-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-
ethylenediamine (17c)
1
Colorless oil. Yield: 85%. H NMR (400 MHz, Chloroform-d)
δ 7.55–7.49 (m, 2H, -ArH), 7.07–7.01
(m, 2H, -ArH), 4.64 (p, J = 6.1 Hz, 1H, -OCH(CH3)2), 4.09 (s, 2H, -NH2), 3.39 (d, J = 16.0 Hz, 2H, -CH2-),
2.91 (d, J = 17.3 Hz, 2H, -CH2-), 2.86 (s, 3H, -CH3), 2.14 (s, 2H, -CH2-), 1.44 (s, 9H, -C(CH3)3), 1.41 (s, 3H,
-CH3), 1.40 (s, 3H, -CH3).
N-tert-Butyloxycarbonyl-N’-(1-(4-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-ethylenediamine (17d
)
1
Colorless oil. Yield: 85%. H NMR (400 MHz, Chloroform-d)
δ 7.48 (d, J = 8.5 Hz, 2H, -ArH),
7.11–7.01 (m, 2H, -ArH), 4.64 (h, J = 6.0 Hz, 1H, -OCH(CH3)2), 4.19 (s, 2H, -CH2-), 3.33 (d, J = 5.9 Hz,
2H, -CH2-), 2.98 (s, 2H, -CH2-), 1.45 (s, 9H, -C(CH3)3), 1.42 (d, J = 1.3 Hz, 3H, -CH3), 1.40 (d, J = 1.4 Hz,
3H, -CH3).
N-tert-Butyloxycarbonylaminoethyl-N’-(1-(4-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-
piperazine (17e)
1
Colorless oil. Yield: 75%. H NMR (400 MHz, DMSO-d6)
δ 7.65–7.60 (m, 2H, -ArH), 7.17–7.10 (m,
2H, -ArH), 6.63 (t, J = 5.7 Hz, 1H, -NH-), 4.74 (hept, J = 6.0 Hz, 1H, -OCH(CH3)2), 3.77 (s, 2H, -CH2-),
2.98 (q, J = 6.4 Hz, 2H, -CH2-), 2.43–2.15 (m, 10H, -CH2-), 1.37 (s, 9H, -C(CH3)3), 1.32 (s, 3H, -CH3), 1.31
(s, 3H, -CH3).
General procedure for the preparation of compounds 18a–e.
Compounds 17a–e (0.51 mmol) was added to saturated hydrochloric acid ethanol solution (10
mL) at room temperature and stirred for 30 min. The reaction solution was filtered and washed with
ethanol (20 mL) to give 18a–e.

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N-(1-(4-Isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-N-methyl-ethylenediamine hydrochloride (18a
)
White solid. Yield: 95%. MP: 186.5–187.7 ◦C. 1H NMR (400 MHz, Deuterium Oxide)
δ
7.46–7.38
(m, 2H, -ArH), 7.13–7.07 (m, 2H, -ArH), 4.69–4.65 (m, 1H, -OCH(CH3)2), 4.41 (s, 2H, -CH2-), 3.14 (td, J =
6.3, 1.3 Hz, 2H, -CH2-), 3.06 (td, J = 6.3, 1.3 Hz, 2H, -CH2-), 2.49 (s, 3H, -CH3), 1.26 (s, 3H, -CH3), 1.25
(s,3H, -CH3). 13C NMR (101 MHz, Deuterium Oxide)
δ 150.5, 126.7, 125.0, 117.1, 71.9, 52.9, 49.0, 41.2,
35.2, 20.9. HRMS (ESI) m/z [M+H]+ Calcd for C14H23N6O+: 291.19279. Found: 291.19278.
N-(1-(4-Isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-N-methyl-1,3-propylenediamine
hydrochloride (18b)
Colorless oil. Yield: 92%. 1H NMR (400 MHz, Deuterium Oxide)
δ 7.42–7.36 (m, 2H, -ArH),
7.12–7.07 (m, 2H, -ArH), 4.69–4.63 (m, 1H, -OCH(CH3)2), 4.72 (s, 2H, -CH2-), 3.32–3.24 (m, 2H, -CH2-),
2.99–2.90 (m, 2H, -CH2-), 2.86 (s, 3H, -CH3), 2.04 (tt, J = 9.8, 6.6 Hz, 2H, -CH2-), 1.25 (s, 3H, -CH3), 1.23
(s, 3H, -CH3). 13C NMR (101 MHz, Deuterium Oxide)
δ
159.4, 147.9, 126.7, 124.5, 117.2, 71.9, 53.7, 48.1,
41.1, 36.2, 22.0, 20.9. HRMS (ESI) m/z [M+H]+ Calcd for C15H25N6O+: 305.20844. Found: 305.20840.
N-Methyl-N’-(1-(4-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-ethylenediamine hydrochloride (18c
White solid. Yield: 92%. MP: 179.7–180.7 ◦C. 1H NMR (400 MHz, Deuterium Oxide)
7.43–7.36
)
δ
(m, 2H, -ArH), 7.12–7.06 (m, 2H, -ArH), 4.67 (d, J = 6.1 Hz, 1H, -OCH(CH3)2), 4.49 (s, 2H, -CH2-), 3.37
(ddd, J = 7.3, 6.1, 1.5 Hz, 2H, -CH2-), 3.29 (ddd, J = 7.7, 6.1, 1.6 Hz, 2H, -CH2-), 2.65 (s, 3H, -CH3), 1.25 (s,
3H, -CH3), 1.24 (s, 3H, -CH3). 13C NMR (101 MHz, Deuterium Oxide)
δ 159.2, 149.3, 126.4, 124.7, 117.2,
71.9, 44.5, 43.4, 40.7, 33.1, 20.9. HRMS (ESI) m/z [M+H]+ Calcd for C14H23N6O+: 291.19279. Found:
291.19269.
N-(1-(4-Isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-ethylenediaminehydrochloride (18d)
White solid. Yield: 93%. MP: 183.1–183.9 ◦C. 1H NMR (400 MHz, Deuterium Oxide)
δ 7.43–7.38
(m, 2H, -ArH), 7.13–7.08 (m, 2H, -ArH), 4.69–4.65 (m, 1H, -OCH(CH3)2), 4.52 (s, 2H, -CH2-), 3.37 (dd,
J = 7.2, 5.9 Hz, 2H, -CH2-), 3.26 (ddd, J = 7.4, 6.3, 1.1 Hz, 2H, -CH2-), 1.26 (s, 3H, -CH3), 1.25 (s, 3H,
-CH3). 13C NMR (101 MHz, Deuterium Oxide)
δ 159.2, 149.3, 126.4, 124.7, 117.2, 71.9, 44.6, 40.7, 35.5,
20.9. HRMS (ESI) m/z [M+H]+ Calcd for C13H21N6O+: 277.17714. Found: 291.17709.
N-aminoethyl-N’-(1-(4-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-piperazinehydrochloride (18e)
Yellow solid. Yield: 92%. MP: 183.2–183.9 ◦C. 1H NMR (400 MHz, Deuterium Oxide)
δ
7.46–7.40
(m, 2H, -ArH), 7.11–7.05 (m, 2H, -ArH), 4.00 (s, 2H, -CH2-), 3.35–3.26 (m, 4H, -CH2-), 3.19 (s, 4H, -CH2-),
2.82 (s, 4H, -CH2-), 1.25 (s, 3H, -CH3), 1.23 (s, 3H, -CH3). 13C NMR (101 MHz, Deuterium Oxide)
δ
158.9, 151.7, 126.7, 116.9, 71.9, 52.5, 51.8, 49.3, 48.3, 33.7, 20.9. HRMS (ESI) m/z [M+H]+ Calcd for
C17H28N7O+: 346.23499. Found: 346.23494.
N-2,3-bis-(tert-Butoxycarbonyl)guanidino-N’-(1-(4-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-
N’-methyl-ethylenediamine (19)
To a solution of 18a (0.26g, 0.80mmol) in dry DMF was added to stirred for 10 min and then
1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (0.23g, 0.80mmol) was added and stirred for
5 h. The reaction mixture was quenched with water and extracted with ethyl acetate. The organic
layer was dried over Na2SO4, and the solvent removed under reduced pressure. The solid residue
thus obtained was purified by column chromatography on silica gel using EtOAc/petroleum (1/2,
1
V/V) as eluent to give 19 as yellow oil (0.22g, 82%). H NMR (400 MHz, Chloroform-d)
δ 11.45 (s, 1H,
-NH-), 8.48 (s, 1H, -NH-), 7.65–7.59 (m, 2H, -ArH), 7.06–6.99 (m, 2H, -ArH), 4.63 (p, J = 6.1 Hz, 1H,
-OCH(CH3)2), 3.79 (s, 2H, -CH2-), 3.52 (q, J = 5.5 Hz, 2H, -CH2-), 2.78 (t, J = 6.0 Hz, 2H, -CH2-), 2.39 (s,
3H, -CH3), 1.53 (s, 3H, -CH3), 1.52 (s, 9H, -C(CH3)3), 1.41 (s, 9H, -C(CH3)3), 1.39 (s, 3H, -CH3).
N-Guanidino-N’-(1-(4-isopropoxyphenyl)-1H-tetrazol-5-yl-methyl)-N’-methyl-ethylenediamine
hydrochloride (20)
Compounds 19 (0.55 mmol) was added to saturated hydrochloric acid ethanol solution (10 mL) at
room temperature and stirred for 30min. The reaction solution was filtered and washed with ethanol
1
(20 mL) to give 20. Yellow oil. Yield: 92%. H NMR (400 MHz, Deuterium Oxide)
δ
7.40 (dd, J = 8.7,
5.3 Hz, 2H, -ArH), 7.10 (dd, J = 8.7, 4.7 Hz, 2H, -ArH), 4.76 (s, 1H, -NH-), 4.66 (d, J = 5.0 Hz, 2H, -CH2-),
3.59 (dt, J = 25.7, 6.0 Hz, 2H, -CH2-), 3.42 (dd, J = 14.0, 7.2 Hz, 2H, -CH2-), 2.87 (d, J = 34.7 Hz, 3H, -NH-,

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-NH2), 1.24 (d, J = 6.0 Hz, 6H, -CH3). 13C NMR (101 MHz, Deuterium Oxide)
δ
159.4, 148.1, 126.7, 124.5,
117.2, 71.9, 54.5, 48.4, 41.6, 36.3, 29.5, 20.9. HRMS (ESI) m/z [M+H]+ Calcd for C15H25N8O+: 333.21458.
Found: 333.21451.
3.3. Radioactive Methylation Assay and MOA Study
The enzyme inhibitory activities were measured by the radioisotope assay in ShangHai
Chempartner Co. Ltd. according to the standard protocol. The similar procedure was recently
performed in the publication [25] The radioactive methylation assay was performed in 1 assay buffer
×
(modified Tris buffer) system containing enzyme (PRMT1/5), peptide and [3H]-SAM solution and
compounds on the assay plate. After 250 nL of compound solutions were added to the assay plate,
15
prepared compound stock plates and the whole system (the final concentration of PRMT1 or PRMT5
was 0.5 nM or 2 nM in the system) incubated for 15 min at room temperature. Then, 10 L of peptide
and (3H)-SAM mixed solution was added to each well to start the reaction (the final concentration
of (3H)-SAM was 0.25
M in the system) and the reaction incubated for 60 min at room temperature.
Afterwards, the reaction was stopped with addition of 5 L cold SAM solution to per well. Then, 25
µL PRMT1/5 enzyme solution or 1
×
assay buffer for negative control was transfer to per well of
µ
µ
µ
µL
of volume per well was transferred to Flashplate from assay plate and incubated for 1 h minimum at
room temperature. Finally, the Flashplate was washed with dH2O and 0.1% Tween-20 three times and
then reading plate in Mi crobeta using program 3H-Flashplate. The data was analyzed in GraphPad
Prism 5 to obtain IC50 values.
The MOA study of compound 9a was performed against PRMT1 with respect to SAM and peptide
substrate independently. In brief, the peptide concentration was kept at 100nM (10
values of 9a were determined at different SAM concentrations (0.5, 1, 2, 6, 20 and 60
the SAM concentration was kept at 250 nm (1 Km) and the IC50 values of 9a monitored at different
Km). The method is consistent with the procedure
×
Km) and the IC50
×
Km). And then,
×
peptide concentration (0.5, 1, 3, 10, 30 and 100
described above.
×
3.4. Molecular Modeling
Since the crystal structure of human PRMT1 (hPRMT1) has not been solved, the homology model
of hPRMT1 in complex with SAH, which was built by referencing the crystal structure of rat PRMT3
(rPRMT3, PDB 1F3L) as the template according to our previous publication [23], was used for the
current molecular modeling study. By overlaying the crystal structures of PRMT6-MS023 [PDB ID
5E8R] with hPRMT1, the position of MS023 was used as the reference to indicate the binding site,
and then compound 9a was docked by using glide module in Schrödinger Release 2017-4 with default
settings [33].
The compound 9a and hPRMT1-SAH complex was then prepared for molecular dynamic
simulations. QM calculations were performed by using the B3LYP 6-31G* basis set within
Gaussian16 [34] to optimize the molecular geometries of compound 9a and SAH, and the atom-centered
point charges were calculated to fit the electrostatic potential using RESP [35]. The system was explicitly
solvated in a truncated octahedral TIP3P water box (12 Å from the complex to avoid periodic artefacts
from occurring) by using Amber 16 with the amber ff14SB force field [36] and the Generalized Amber
Force field (GAFF) [37]. 11 K+ ions were added to neutralize the charges of the system. The whole
system was first optimised by energy minimisations and equilibrations according to our standard
protocol [23], and then 100 ns free production molecular dynamic simulation was followed in the NPT
ensemble (T = 300 K; P = 1 atm). The long-range electrostatic effects were treated by using Periodic
boundary conditions (PBC) and particle-mesh-Ewald method (PME), and the temperature was coupled
to an external bath using a weak coupling algorithm [38]. The non-bonded interaction cutoff was set as
8 Å and the bond interactions involving H-atoms were constrained by using the SHAKE algorithm.
The time step to solve the Newton’s equations was chosen as 2 fs and the trajectory files were collected
every 100 ps for the subsequent analysis.

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MM-PBSA and Normal Mode were performed for the binding free energy calculations, based
on 300 snapshots collected from the stable trajectory. The binding free energy contribution of each
residues was calculated, and only those greater than
interactions analysis.
−
0.7 Kcal/mol were recorded for detailed
3.5. Cell Culture
The MDA-MB-231 cell line used in this study was obtained from the American Type Culture
Collection (ATCC, Rockville, MD, USA), and was grown in dulbecco’s modified eagle medium (DMEM,
Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS)
(Gibco). The cells were incubated at 37 ◦C and 5% CO2.
3.6. Cell Viability Assay
The cytotoxicity was determined by the CCK8 assay. Briefly, MDA-MB-231 cells (5
were seeded in 96-well plates in DMEM containing 10% FBS and grew for 24 h. The exponentially
×
103 cells/well)
growing cells were incubated with various concentrations of compounds for 48h/72h in serum free
DMEM at 37 ◦C (5% CO2, 95% humidity). CCK8 reagent (10
µL) was added to each well and incubated
for further 2 h, and then the absorbance was analyzed in a multiwell-plate reader (BioTek ELx800) at
450 nm.
3.7. Western Blotting
MDA-MB-231 cells were maintained on 6-well plates at the appropriate density. After the
attachment, cells were treated with 9a at indicated concentrations or DMSO control for 48 h. Total cell
lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The blots were
blocked with 5% nonfat milk for 30 min and the target proteins were probed with appropriate specific
antibody overnight at 4 ◦C, respectively. The blots were washed with TBST for three times and then
incubated with anti-rabbit secondary antibody (HRP conjugated) for 1 h. After another three washes,
bands were detected in a ChemiScope3400 imaging system using ECL substrate (Millipore, Burlington,
MS, USA). Primary antibodies used were as follows: anti-PRMT1 (Cell Signaling Technology no.2449,
Danvers, MA, USA), anti-ADMA (Cell Signaling Technology no.13522), anti-SDMA (Cell Signaling
Technology no.13222), anti-GAPDH (Cell Signaling Technology no. 5174), anti-H4R3me2a Abcam,
ab9231), Wnt3a (Cell Signaling Technology no. 2721s), anti-DDAH (Abcam, ab9231, Cambridge, UK),
Wnt5a/b (Cell Signaling Technology no. 2530s),
β-catenin (Cell Signaling Technology no. 8480s),
MMP9 (Cell Signaling Technology no.1366s). The results were obtained from multiple membranes.
No significant differences were observed among loading controls. Therefore, we presented the
representative loading control blot image in the figure.
4. Conclusions
In summary, through computer aided drug design, we designed and synthesized 22 1,5-substituded
tetrazole derivatives. Among them, five compounds (9a
effects on PRMT1. Compound 9a was identified as the most potent PRMT1 inhibitor (IC50 = 3.5
,
9f
,
16c
,
18a
,
18e) showed strong inhibitory
M) in
µ
the current study, and it showed strong selectivity to PRMT1 (type I PRMT) with respect to PRMT5
(type II PRMT). The MOA assay showed that compound 9a did not compete with either SAM or
peptides, but according to the crystal structure of human PRMT6 with its inhibitor and combining with
the molecular dynamic simulation study, it was believed that compound 9a bound to substrate-arginine
binding site. By Western blotting, it was confirmed that compound 9a inhibited the PRMT1 pathway,
and the canonical Wnt/β-catenin signaling pathway was down-regulated. The discovery of compound
9a is likely to prove to be very important for the understanding of PRMT1 function, and is a potential
lead compound for future drug design efforts targeting PRMT1.

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Author Contributions: W.H. and H.W. (Hao Wang) conceived and designed the experiments; Y.S., Z.W., H.Y.,
X.Z., H.W. (Han Wu) and L.M. performed the experiments; W.H., H.W. (Hao Wang) and F.X. analyzed the data;
W.H., H.W. (Han Wu) and Y.S. wrote the paper.
Funding: This work was supported by National Natural Science Foundation of China (81660588, 81773582),
the Key R&D Program of Ningxia (2018BFG02004), and the Program for Leading Talents of Ningxia Province
(KJT2018004).
Acknowledgments: The authors thank Ian Paterson and Xinyi Yang for many helpful suggestions and corrections
on the current manuscript.
Conflicts of Interest: All authors declare no conflict of interest.
Abbreviations
PRMT1
SAM
Protein arginine methyltransferase 1
S-adenosyl-L-methionine
mMA
SAH
ADMA
SDMA
DIBAL-H
STAB
monomethylarginine
S-adenosyl-L-homocysteine
Asymmetrical dimethylargine
Symmetrical dimethylarginine
Diisobutylaluminium hydride
Sodium triacetoxyborohydride
Structure-activity relationship
Mechanism of action
SAR
MOA
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